RESUMEN
Lysine biosynthesis in bacteria and plants commences with a condensation reaction catalysed by dihydrodipicolinate synthase (DHDPS) followed by a reduction reaction catalysed by dihydrodipicolinate reductase (DHDPR). Interestingly, both DHDPS and DHDPR exist as different oligomeric forms in bacteria and plants. DHDPS is primarily a homotetramer in all species, but the architecture of the tetramer differs across kingdoms. DHDPR also exists as a tetramer in bacteria, but has recently been reported to be dimeric in plants. This study aimed to characterise for the first time the structure and function of DHDPS and DHDPR from cyanobacteria, which is an evolutionary important phylum that evolved at the divergence point between bacteria and plants. We cloned, expressed and purified DHDPS and DHDPR from the cyanobacterium Anabaena variabilis. The recombinant enzymes were shown to be folded by circular dichroism spectroscopy, enzymatically active employing the quantitative DHDPS-DHDPR coupled assay, and form tetramers in solution using analytical ultracentrifugation. Crystal structures of DHDPS and DHDPR from A. variabilis were determined at 1.92 Å and 2.83 Å, respectively, and show that both enzymes adopt the canonical bacterial tetrameric architecture. These studies indicate that the quaternary structure of bacterial and plant DHDPS and DHDPR diverged after cyanobacteria evolved.
Asunto(s)
Anabaena variabilis/enzimología , Proteínas Bacterianas/química , Dihidrodipicolinato-Reductasa/química , Hidroliasas/química , Anabaena variabilis/genética , Proteínas Bacterianas/genética , Dicroismo Circular , Cristalografía por Rayos X , Dihidrodipicolinato-Reductasa/genética , Hidroliasas/genética , Estructura Cuaternaria de Proteína , Relación Estructura-ActividadRESUMEN
Here, we review recent studies aimed at defining the importance of quaternary structure to a model oligomeric enzyme, dihydrodipicolinate synthase. This will illustrate the complementary and synergistic outcomes of coupling the techniques of analytical ultracentrifugation with enzyme kinetics, in vitro mutagenesis, macromolecular crystallography, small angle X-ray scattering, and molecular dynamics simulations, to demonstrate the role of subunit self-association in facilitating protein dynamics and enzyme function. This multitechnique approach has yielded new insights into the molecular evolution of protein quaternary structure.